Hyaluronan (HA) is a ubiquitous glucosaminoglycan in the extracellular matrix, shown to play a central role in embyrogenesis, inflammation, wound healing, and tumour metastasis.
(Toole, B. P. (1990) Hyaluronan and its binding proteins, the hyaladherins. Curr. Opin. Cell. Biol. 2: 839-844.
Toole, B. P. (1982). Development role of hyaluronate. Conn. Tiss. Res 10: 93-100.)
Interaction between HA and RHAMM, a receptor for HA-mediated motility are required for motile behaviour of a wide variety of cells including sperm, fibroblasts, astrocytes, microglia and white blood cells.
(Entwistle, J. Zhang, S., Yang, B., Wong, C. Hall, C. L., Curpen, G., Mowat M., Greenberg, A. H., and Turley, E. A. (1995). Cloning and characterization of the gene encoding the hyaluronan receptor RHAMM: the role of a secreted isoform in the regulation of focal adhesion formation. Gene 163: 233-238
Yang, B., Yang, X. Zhang, S., Turley, M., Samuel, S., Savani, R. C., Greenberg, A. H., and Turley, E. A. (1995). Overexpression of the hyaluronan receptor RHAMM is transforming, and is required for H-ras transformation. Cell 8: 19-28.
Masellis-Smith, A., Belch, A. R., Mant, M. J., Turley, E. A., and Pilarski., L. M. (1996). Hyaluronan-dependent motility of B cells and leukemic plasma cells in multiple myeloma: Alternate usage of RHAMM and CD44. Blood 87: 1891-1899.
Turley, E. A., Belch, A. R., Poppema, S., and Pilarski, L. M. (1993). Expression and function of a receptor for hyaluronan-mediated motility (RHAMM) on normal and malignant B lymphocytes. Blood 81: 446-453.
Pilarski, L. M. Miszta, H., and Turley, E. A. (1993). Regulation expression of a receptor for hyaluronan-mediated motility RHAMM) on human thymocytes and T cells. J. Immunol. 150: 4292-4302
S., K. B., McCoshen, J., Kredentser, J., and Turley, E. (1994). The Regulation of Sperm Motility by a Novel Hyaluronan Receptor. Fertility and Sterility 61: 935-940.
Turley, E. A., Sossain, M. Z., Sorokan, T., Jordan, L. M., and Nagy, J. I. (1994) Astrocyte and microglial motility in vitro is functionally dependent on the hyaluronan receptor RHAMM. Glia 12: 68-80)
The cells that populate the blood are all derived from multipotential (or pluripotential) stem cells present in bone marrow. Multipotential stem cells continually proliferate and renew themselves, but also give rise to common progenitor cells. Once committed, progenitor cells differentiate into immature precursor cells of the various blood cell lineages which, following further differentiation stages, eventually give rise to mature functional blood cells, such as erythrocytes, monocytes, lymphocytes, and polymorphonuclear cells. (Golub, E. S., Green, D. R. (1991) Immunology A Synthesis, 2:205; Kuby, J. (1997) Immunology, 3:50; Roitt, I., Brostoff, J., Male, D. (______) Immunology, 4:2.1). Terminally differentiated blood cells generally lose their ability to proliferate—indeed mammalian erythrocytes and platelets contain no nuclei—and thus have finite lifetimes. Granulocytes may exist only for a matter of hours, whereas human erythrocytes remain in circulation for over 100 days. Although some lymphocytes have life-spans measured in years, most are short lived (for example, 3 days—3 weeks). Therefore, to maintain steady-state numbers of particular blood cell types, there must be a continual production of these from the bone marrow. This process is known as haemopoiesis (haematopoiesis) or the haemopoietic process. While much remains to be learned, it is clear that many steps in the haemopoietic process (haemopoiesis) are controlled by certain cytokines (for example, GM-CSF and G-SCF and erythropoietin (EPO)), also known as haemopoietic growth factors, and by microenvironmental factors including stromal cells and extra-cellular matrix components (for example, hyaluronan).
Clinically, the term “mobilization” usually refers to the process whereby cells leave the bone marrow and enter the blood. The mechanism whereby this occurs is not known by those skilled in the art.
However, I believe mobilization can be viewed as the stimulation of de-adhesive behaviour by hematopoietic cells.
I believe that under normal circumstances, hematopoietic cells are anchored in their environment by receptors known as adhesion molecules. These adhesion molecules bind to components of the extracellular and cellular matrix within tissues to anchor the cell, or alternatively, to permit its migratory behaviour. Among the receptors thought to be important are those binding HA.
I believe mobilization involves two events: 1) a release from the anchoring interactions (de-adhesion) and 2) the stimulation of migratory behaviour. To reach the circulation from lymphoid tissue or the bone marrow a cell must “let go” of its anchoring interaction, activate adhesion receptors involved in migration (motile behaviour) and then actually locomote through tissue, penetrate endothelial cell linings and enter the blood vessel (intravasate). HA and receptors for HA are known to be involved in cell migration, motility and de-adhesion.
Most hematopoietic cells are anchored in the bone marrow or other lymphoid tissues. A stimulating/inducing event is required to mobilize them to the circulation as this is an active, not a passive process. Present practice involves administration of a variety of cytokines, often together with chemotherapeutic agents to mobilize hematopoietic cells to the blood. The mechanism for this is unknown. However, stem cell mobilization, the recruitment of hematopoietic stem cells into the blood where they can be easily harvested, is clinically performed using G-CSF and GM-CSF with or without chemotherapy.
(Weaver, C. H., Hazeltonn, B., Birch, R., Palmer, P., Allen, A., Schwwartzberg, L. and West, W. (1995). An analysis of engraftment kinetics as a function of the CD34 content of peripheral blood progenitor cell collections in 692 patients after the administration of myeloablative chemotherapy. Blood 86: 3961-3969.
Boiron, J.-M., Marit, G., Faberes, C., Cony-Makhoul, P., Foures, C., Ferrer, A.-M., Cristol, G., Sarrat, A., Girault, D., and Reiffers, J. (1993) Collection of peripheral blood stem cells in multiple myeloma following single high-dose cyclophosphamide with and without recombinant human gran ulocte-macrophage colony-stimulating factor (rh GM-CSF). Bone Marrow Transplantation 12: 49-55.
Schiller, G., Vescio, R., Freytes, C., Spitzer, G., Sahebi, F., Lee, M. Wu, S.-H., Cao, J., Lee. J. C., Hong, C. H. Lichtenstein, A., Lill, M., Hall, J., Berenson, R., and Berenson, J. (1995) Transplantation of CD34+peripheral blood progenitor cells after high-dose chemotherapy for patients with advanced multiple myeloma. Blood 86: 390-397.)
Mobilized peripheral blood stem cell collections (PBSC) resulting from the use of G-CSF or GM-CSF are transplanted into, e.g. a cancer patient. These can be either the total population of the mobilized white blood cells or purified stem cells. Stem cells are those cells able to reconstitute the hematopoietic system of an organism, which requires self renewal of stem cells as well as differentiation to cells of the various hematopoietic lineages. The CD34 marker is characteristic of stem cells. Other cells that are mobilized include polymorphonuclear white blood cells (cells that mediate inflammation and clearance of pathogens), mononuclear white blood cells (lymphocytes and monocytes) and red blood cell progenitors (erythroblasts).
Mobilization of CD34+ stem cells is a rapidly expanding clinical technique for obtaining material for autologous or allogeneic hematopoietic transplantation. Mobilization of polymorphs is a valuable adjunct to heavy chemotherapy to maintain innate defense mechanisms. Currently, both methods rely on mobilization by growth factors (G-CSF or GM-CSF) which is expensive, causes bone pain, and has unknown side effects for normal donors. It takes up to about 4 weeks of treatments to collect sufficient material for a transplant. After growth factor treatment, CD34+ cells reach a maximum level of 2-4% in blood.
The mature cells of the haemopoietic system include erythrocytes, polymorphonuclear-cells (PMN), lymphocytes, monocytes, macrophages, osteoblasts, osteoclasts, mast cells, and platelets. These all have a limited life-span, and must be replaced as they die. To achieve a balance between cell death and renewal, the bone marrow must not only continuously provide progenitor cells, but also control the commitment of these to the various lineages so that the correct proportions of mature cells are produced. The basic control mechanisms, especially of the earliest stages of haemopoiesis, are as yet poorly understood. There appears to be some compartmentalization of the marrow, and microscopic ‘nests’ of particular precursor cells have been identified. However, it has been shown that the survival and proliferation of stem and progenitor cells is dependent upon the presence of accessory cells which in vitro form into an adherent ‘stromal’ layer. In the absence of the stromal layer, stem and progenitor cells die and so it appears the stromal cells support proliferation and differentiation by intercellular interactions including production of growth factors into the extracellular milieu. In culture, stromal cells have been shown to produce GM-CSF, M-CSF, and a megakaryocyte-colony stimulating factor (or molecules functionally equivalent to these). It is widely believed that such growth factors (cytokines) are involved in haemopoiesis, but their exact role(s) in self-renewal of stem cells, differentiation of stem cells into common progenitor cells, and the proliferation and differentiation of committed progenitor cells, remains unclear. More definite roles of these cytokines in the growth stimulation and development of later-stage precursors have been evinced by the use of in vitro colony-forming culture systems introduced by Metcalf and colleagues in the 1970s. In these experimental systems multipotential stem cells, progenitors, or precursors are suspended in the absence of stromal cells in semi-solid agar growth medium. Without the addition of exogenous cytokines, the cells die. However, they can be stimulated to grow, multiply, and differentiate to form colonies of various blood cell lineages by adding into the growth medium dilutions of certain supernatants obtained from activated leukocytes or by addition of the now readily available purified recombinant cytokines including GM-CSF. Furthermore, injection of recombinant cytokines into experimental animals, and into patients in clinical trials to assess therapeutic potential of individual cytokine products, has shown that IL-3, GM-CSF, and G-CSF stimulate the production of white cells such as granulocytes and monocytes, thus lending support for physiological roles of such cytokines. In addition, it has also become apparent that these cytokines not only support the growth and differentiation of immature blood cells, but also in many instances are effector molecules for the functional activation of mature cells.
The molecular cloning of both murine and human homologues of IL-3, GM-CSF, G-CSF, M-CSF, IL-5, and EPO has been accomplished.
Of the four ‘granulocyte-macrophage’ CSFs, GM-CSF was the first to be isolated and characterized. GM-CSF was shown to induce the proliferation of murine bone marrow—or spleen-derived haemopoietic cells containing granulocyte and macrophage progenitors giving rise to colonies containing mainly granulocyte and macrophage precursors. In this respect, GM-CSF appears to share biological properties with the subsequently characterized IL-3. However, more recent studies suggest that GM-CSF acts on ‘later-stage’ multipotential cells than IL-3. Also, GM-CSF appears to be less active than IL-3 in stimulating the proliferation of erythroid and megakaryocytic precursors. Nevertheless, like IL-3, GM-CSF can be shown to have activities in mature cells of the granulocyte and macrophage lineages.
GM-CSF (Granulocyte-Macrophage Colony Stimulating Factor) acts directly and selectively on granulocyte/macrophage progenitors to stimulate growth and differentiation in vitro of cells belonging to these lineages, e.g. neutrophils, eosinophils, macrophages. These pleiotropic activities have also been demonstrated for recombinant GM-CSF. Besides regulation of the proliferation and differentiation of the progenitor/precursor cells of the myeloid lineage, GM-CSF has also been shown to activate the functions of mature myeloid cell types. For example, GM-CSF has been found to induce macrophage tumoricidal activity against the malignant melanoma cell line, A375. IFNγ can also behave as a macrophage activating factor, but in contrast to GM-CSF requires an additional secondary stimulus, e.g. bacterial LPS, to evoke tumoricidal activity. In addition, GM-CSF activates macrophages to inhibit the replication of Trypanosoma cruzi (a unicellular parasite that is the aetiological agent of Chagas disease, or American trypanosomiasis) and increases respiratory oxidative processes. Furthermore, the replication of HIV-1 in the human monocytic cell line U937 has been shown to be moderately inhibited by GM-CSF, and more effectively by the combination of GM-CSF and IFNγ. These results suggest that GM-CSF could have a potential physiological role in eosinophils and macrophage activation and thus possibly could be used prophylactically or therapeutically against a range of microbial agents that replicate in macrophages.
In neutrophils and eosinophils, GM-CSF stimulates a number of functions. In particular, GM-CSF enhances phagocytosis of bacteria and yeasts by neutrophils. Purified recombinant human GM-CSF has also been shown to enhance the cytotoxic activity of neutrophils and eosinophils against antibody-coated target cells. These observations and others in which the anti-microbial functions of neutrophils and eosinophils are increased by GM-CSF, strongly suggest an important role for this mediator in host defence.
When mice are repeatedly injected intraperitoneally with recombinant murine GM-CSF, there is a rapid and sustained increase in the number and functional activity of peritoneal macrophages, granulocytes (neutrophils and eosinophils) as well as increased numbers of circulating monocytes. (GM-CSF usually takes about two weeks to act.) Marked increases in neutrophil, eosinophil, and monocyte numbers have also been observed following injection of recombinant human GM-CSF into AIDS patients and non-human primates. However, there may be complications associated with GM-CSF therapy. Metcalf and colleagues have shown that transgenic mice containing a constitutively expressed murine GM-CSF gene have pathological lesions soon after birth in various tissues, including lens, retina, and striated muscle, resulting from activated-macrophage infiltration. Thus, chronic macrophage activation in GM-CSF therapeutic schedules should be avoided. (Activated macrophages are known to produce a number of inflammatory mediators including cytokines such as TNFα and IL-1 which may induce tissue damage.)
In contrast to its growth-stimulating effects, GM-CSF can act as a differentiation factor. Its actions on mature macrophages and neutrophils, for example, might be considered as consequences of its differentiation-inducing capacity. One way to limit the proliferation of tumour cells is to decouple growth-factor-driven self-renewal from growth-factor-induced differentiation. In other words, the more ‘differentiated’ tumour cells become, the less able they are to multiply. In this regard, GM-CSF has been shown to induce differentiation of the myeloid leukaemic cell line HL60 and suppress its self-renewal. However, in several other studies, GM-CSF stimulated the proliferation of HL60 cells. Differentiation can be monitored by measuring expression of various plasma membrane-associated antigens, e.g. CD14 (monocyte/macrophage marker) and CD57 (NK cell marker). These have been reported to be induced by GM-CSF in small cell lung cancer (SCLC) cell lines, suggesting that SCLC has a myeloid cell origin. This would be consistent with a proposal that SCLC arises from macrophage precursors which infiltrate damaged lung tissues, such as occur in heavy smokers. The ready availability of recombinant human GM-CSF and the limited distribution of GM-CSF receptors to cells of the myeloid and possibly erythroid lineages may thus help to define the histological origin of tumours, and suggests alternative therapeutic modalities for the treatment of cancers such as SCLC.
It thus appears that while the use of Granulocyte-macrophage colony stimulating factor (GM-CSF) has been used as a stimulant for the production of stem cells, progenitor cells, precursor cells, accessory cells and macrophages there are a substantial number of disadvantages in its use, those discussed above and the appearance of bone pain, fever, myalgia and erythema in patients to whom cytokines such as GM-CSF and G-CSF were administered, which make the use of GM-CSF-and G-CSF not as desirable.
It is therefore an object of this invention to provide the use of another and other compounds which provide similar effects as GM-CSF and G-CSF but with lesser side effects.
It is a further object of this invention to provide such compounds in suitable dosages for effective and safe use.
It is still a further object of this invention to provide, improved treatments and regimens of treatment.
It is a further object of the invention to provide a novel use for hyaluronan (HA) for mobilizing cells such as hematopoietic cells.
Further and other objects of the invention will be realized by those skilled in the art from the following summary of invention and detailed description of embodiments thereof.